Purification and characterization of equine relaxin.

The research article describes the process of purifying and characterizing equine relaxin, a hormone produced by the placenta during pregnancy, from horse placentas.

Materials

• The researchers used whole horse placentas obtained post-foaling as their source of equine relaxin.

Process of Purification

• The placentas were ground up and extracted using 0.5 N HCl-85% acetone.
• Relaxin was precipitated by increasing acetone concentration to 97%.
• The relaxin was further purified through multiple methods including stepwise elution ion exchange, gel filtration, gradient elution ion exchange chromatographies, and trichloroacetic acid precipitation.

Identification and Characterization

• The resultant equine relaxin was identified to be heterogeneous with one major form (R-1) and several minor forms (R-2 and R-3).
• The purity of the isolated relaxin was assessed through slab and disc gel electrophoresis and dansyl end group analysis.
• Around 1.5 mg of R-1 was obtained per kilogram of placenta.

Potency

• The purified equine relaxin had a potency of 28 U/mg as measured in the mouse inter-pubic ligament bioassay. The dose-response curve was parallel with that of porcine relaxin confirming its biological activity.

Amino Acid Analysis

• An amino acid analysis indicated the presence of tyrosine, histidine, and proline, which are absent in porcine relaxin. These could potentially be unique markers for the identification of equine relaxin.
• The analysis also showed that N-terminal groups were blocked on the major form (R-1) and the presence of the amino acid Lys on one of the minor forms (R-3).

Molecular Weight

• Through electrophoretic migration of relaxin reduced with 2-mercaptoethanol, it was suggested that equine relaxin is comprised of two chains hence it has a lower molecular weight.